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Elucidation of the structure of the ribosome has stimulated numerous proposals for the roles of specific rRNA elements, including the universally conserved helix 69 (H69) of 23S rRNA, which forms intersubunit bridge B2a and contacts the D stems of A- and P-site tRNAs. H69 has been proposed to be involved not only in subunit association and tRNA binding but also in initiation, translocation, translational accuracy, the peptidyl transferase reaction, and ribosome recycling. Consistent with such proposals, deletion of H69 confers a dominant lethal phenotype. Remarkably, in vitro assays show that affinity-purified Deltah69 ribosomes have normal translational accuracy, synthesize a full-length protein from a natural mRNA template, and support EF-G-dependent translocation at wild-type rates. However, Deltah69 50S subunits are unable to associate with 30S subunits in the absence of tRNA, are defective in RF1-catalyzed peptide release, and can be recycled in the absence of RRF.

Translocation of the tRNA-mRNA complex is a fundamental step in the elongation cycle of protein synthesis. Our studies show that the ribosome can translocate a P-site-bound tRNA Met with a break in the phosphodiester backbone between positions 56 and 57 in the T⌿C-loop. We have used this fragmented P-site-bound tRNA Met to identify two 2 -hydroxyl groups at positions 71 and 76 in the 3 -acceptor arm that are essential for translocation. Crystallographic data show that the 2 -hydroxyl group at positions 71 and 76 contacts the backbone of 23S rRNA residues 1892 and 2433-2434, respectively, in the ribosomal E site. These results establish a set of functional interactions between P-site tRNA and 23S rRNA that are essential for translocation.R ibosomes are the macromolecular complexes responsible for protein synthesis. Escherichia coli ribosomes are made up of a large 50S subunit and a small 30S subunit that contain three tRNA-binding sites (A, aminoacyl; P, peptidyl; E, exit). One of the fundamental steps in the elongation cycle of protein synthesis is the step-wise movement-called translocation-of tRNAs from one site to the next within the ribosome. Very little is known about how this highly precise and complex process occurs on the ribosome. Biochemical (1, 2) and biophysical (3) studies revealed that translocation occurs in two steps. In the first step, after peptide-bond formation, the acceptor ends of deacylated tRNA and peptidyl-tRNA move from P to E, and from A to P sites, respectively, in the 50S subunit, whereas their anticodon ends still interact with the 30S subunit P and A sites, respectively. This movement occurs spontaneously after peptide-bond formation, which results in tRNAs occupying A͞P and P͞E hybrid states. In the second step, elongation factor G (EF-G) catalyzes the movement of the anticodon ends of both tRNAs relative to the 30S subunit, translocating deacylated tRNA from P͞E to E state and peptidyl-tRNA from A͞P to P͞P state. Thus, movement of the acceptor end of tRNAs occurs independently of the anticodon ends, relative to the two ribosomal subunits.EF-G uses the chemical energy of GTP hydrolysis to accelerate translocation of the tRNA-mRNA complex (4). Interestingly, ribosomes can perform spontaneous translocation in the absence of EF-G⅐GTP (5, 6), suggesting that the mechanism of translocation is inherent to the ribosome. Additionally, translocation can occur in the absence of mRNA (7,8), demonstrating that neither codon-anticodon interactions nor mRNA-ribosome interactions are essential for translocation. Thus, the only components indispensable for translocation are the A-and P-site tRNAs and the ribosome.The requirement for peptidyl-tRNA in the A site can be satisfied by a 15-nucleotide anticodon stem-loop analog (ASL)-4 of tRNA (9). The ASL, consisting of a seven-nucleotide loop and a four base-pair stem, translocates, indicating that the D stem, T stem, and acceptor arm of the A-site tRNA are not essential for translocation. In contrast to the ribosomal A site, an ASL bound to the ribo...

The coupled movement of tRNA-mRNA complex through the ribosome is a fundamental step during the protein elongation process. We demonstrate that the ribosome will translocate a P-site-bound tRNA Met with a break in the phosphodiester backbone between positions 17 and 18 in the D-loop. Crystallographic data showed that the acceptor arms of P-and E-site tRNA interact extensively with the ribosomal large subunit. Therefore, we used this fragmented P-site-bound tRNA Met to investigate the contributions of single 2¢ -hydroxyl groups in the 5¢ strand of the acceptor arm for translocation into the ribosomal E-site. EF-G-dependent translocation of the tRNAs was monitored using a toeprinting assay and a fluorescence-based rapid kinetic method. Surprisingly, our results show that none of the 2¢ -hydroxyl groups in the 5¢ strand of the acceptor arm of P-site-bound tRNA Met between positions 1-17 play a critical role during translocation. This suggests that either these 2¢ -hydroxyl groups are not important for translocation or they are redundant and the three-dimensional shape of the P-site tRNA is more important for translocation.

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